Fundamental aspects of eukaryotic biology are controlled by ubiquitylation. Ultimately, the work described here will lead to understanding specificity of the ubiquitylation process. This will lead to greater understanding on a molecular level of the control of a variety of significant biological processes, including cell cycle progression, endocytosis, degradation of misfolded proteins, gene expression, and DMA damage repair. This proposal looks specifically at a functional subclass of enzymes that includes a crucial determinant of DNA damage repair. The specific aims are: (1) To characterize the activity of CHARON (an Arabidopsis protein recently found to enhance ubiquitylation in vitro) in enhancing ubiquitin chain formation. (2) Determine the role of conserved serine and alanine residues in the activity of autocatalytic E2s, including the DNA damage repair protein Rad6p. For the second aim, the ability of CHARON to enhance ubiquitylation will be assessed with a panel of E2 enzymes. Ubiquitylation will be measured by SDS-PAGE Western blotting. In addition, if necessary, the rate of ubiquitylation will be determined by measuring the rate of incorporation of radiolabeled ubiquitin onto substrate protein. Its ability to bind to ubiquitin pathways component will be assessed using a pull down assay. Its ability to form various ubiquitin chain linkages will be assessed using a variety of ubiquitin mutants, and the phenotype of a CHARON knockout will be determined. For the second aim, the conserved residues will be mutated, and wild type and mutant proteins will be assessed for activity in vitro. The in vitro assays mostly involve the analysis of ubiquitylation products using SDS-PAGE Western blot techniques, through development of a novel activity assay is also proposed. Additionally complementation assays will be done in vivo using wild type and mutant proteins expressed in S. cerevisiae strains that have deletions of the protein under study. Expression of wild type, but not mutant, proteins is expected to complement knock out phenotypes. Relevance to public health: Many important functions in human cells are controlled by a process called "ubiquitylation." Certain diseases, such as some forms of cancer and certain neurological diseases are the result of this process not working correctly. This work aims to understand how this process is controlled in healthy cells, with the hope that understanding the process may eventually lead to preventing or curing these diseases. [unreadable] [unreadable] [unreadable]